The RGBarrier assay: the parallel study of gene regulatory element performance at defined chromosomal locations

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

Degradation of compounds present in cork boiling water by gamma radiation

Cork boiling water is an aqueous and complex dark liquor with high concentration of phenolic compounds such as phenolic acids and tannins [1, 2], which are considered biorecalcitrants [2]. Ionizing radiation has been widely studied as an alternative technology for the degradation of organic contaminants without the addition of any other (e.g.: Fenton technologies). The aim of this work was to identify the compounds present in cork boiling water and further evaluate the resulting stable degradation products after gamma irradiation. The irradiation experiments of standard solutions were carried out at room temperature using a Co-60 experimental equipment. The applied absorbed doses were 20 and 50 kGy at a dose rate of 1.5 kGy/h, determined by routine dosimeters [3]. The identification of radiolytic products was carried out by HPLC-DAD-ESI/MS. The phenolic compounds were identified by comparing their retention times and UV–vis and mass spectra with those obtained from standard compounds, when available, as well as by comparing the obtained information with available data reported in the literature. Concerning the obtained results and the literature review, the main cork wastewater components are: quinic, gallic, protocatechuic, vanillic, syringic and ellagic acids. Based on this, we used protocatechuic, vanillic and syringic acids as model compounds to study their degradation by gamma radiation in order to identify the corresponding radiolytic products. Standard aqueous solutions were irradiated and the derivatives of each model compound are represented in figure 1. The obtained results seem to demonstrate that the derivatives of the parent compounds could also be phenolic acids, since it was observed the loss of 44 u (CO2) from the [M-H]- ions. Gallic and protocatechuic acids are identified as derivatives of vanillic and syringic acids, and gallic acid as a protocatechuic acid derivative. Compound 5 ([M-H]- at m/z 169) was tentatively identified as 2,4,6-trihydroxybenzoic acid, since its fragmentation pattern (m/z 151, 125 and 107) is similar to that previously reported in literature [4]. The structure of compound 7 was proposed based on the molecular ion and its fragmentation and compound 6 remains unknown.

INVESTIGATIONS INTO MECHANISMS UNDERLYING EXTREME WAVE FORMATIONS AND COMPUTATIONALLY INTENSIVE SIMULATIONS

Various mechanisms have been proposed to explain extreme waves or rogue waves in an oceanic environment including directional focusing, dispersive focusing, wave-current interaction, and nonlinear modulational instability. The Benjamin-Feir instability (nonlinear modulational instability), however, is considered to be one of the primary mechanisms for rogue-wave occurrence. The nonlinear Schrodinger equation is a well-established approximate model based on the same assumptions as required for the derivation of the Benjamin-Feir theory. Solutions of the nonlinear Schrodinger equation, including new rogue-wave type solutions are presented in the author's dissertation work. The solutions are obtained by using a predictive eigenvalue map based predictor-corrector procedure developed by the author. Features of the predictive map are explored and the influences of certain parameter variations are investigated. The solutions are rescaled to match the length scales of waves generated in a wave tank. Based on the information provided by the map and the details of physical scaling, a framework is developed that can serve as a basis for experimental investigations into a variety of extreme waves as well localizations in wave fields. To derive further fundamental insights into the complexity of extreme wave conditions, Smoothed Particle Hydrodynamics (SPH) simulations are carried out on an advanced Graphic Processing Unit (GPU) based parallel computational platform. Free surface gravity wave simulations have successfully characterized water-wave dispersion in the SPH model while demonstrating extreme energy focusing and wave growth in both linear and nonlinear regimes. A virtual wave tank is simulated wherein wave motions can be excited from either side. Focusing of several wave trains and isolated waves has been simulated. With properly chosen parameters, dispersion effects are observed causing a chirped wave train to focus and exhibit growth. By using the insights derived from the study of the nonlinear Schrodinger equation, modulational instability or self-focusing has been induced in a numerical wave tank and studied through several numerical simulations. Due to the inherent dissipative nature of SPH models, simulating persistent progressive waves can be problematic. This issue has been addressed and an observation-based solution has been provided. The efficacy of SPH in modeling wave focusing can be critical to further our understanding and predicting extreme wave phenomena through simulations. A deeper understanding of the mechanisms underlying extreme energy localization phenomena can help facilitate energy harnessing and serve as a basis to predict and mitigate the impact of energy focusing.

Interactions of pyridinium, pyrrolidinium or piperidinium based ionic liquids with water: measurements and COSMO-RS modelling

Looking for a better knowledge concerning water and ionic liquids (ILs) interactions, a systematic study of the activity coefficients of water in pyridinium, pyrrolidinium and piperidinium-based ILs at 298.2 K is here presented based on water activity measurements. Additionally, the study of the structural effects of the pyridinium-based cation is also pursued. The results show that non-aromatic ILs are interacting more with water than aromatic ones, and among the ortho, meta and para isomers of 1-butyl-methylpyridinium chloride, the ortho position confers a more hydrophilic character to that specific IL. The physicalchemistry of the solutions was interpreted based on dissociation constants, natural bond orbitals and excess enthalpies providing a sound basis for the interpretation of the experimental observations. These results show that hydrogen bonding controls the behavior of these systems, being the anion-water one of the most relevant interactions, but modulated by the anionecation interactions.

Multiscale Modeling and Simulation of Stepped Crystal Surfaces

A primary goal of this dissertation is to understand the links between mathematical models that describe crystal surfaces at three fundamental length scales: The scale of individual atoms, the scale of collections of atoms forming crystal defects, and macroscopic scale. Characterizing connections between different classes of models is a critical task for gaining insight into the physics they describe, a long-standing objective in applied analysis, and also highly relevant in engineering applications. The key concept I use in each problem addressed in this thesis is coarse graining, which is a strategy for connecting fine representations or models with coarser representations. Often this idea is invoked to reduce a large discrete system to an appropriate continuum description, e.g. individual particles are represented by a continuous density. While there is no general theory of coarse graining, one closely related mathematical approach is asymptotic analysis, i.e. the description of limiting behavior as some parameter becomes very large or very small. In the case of crystalline solids, it is natural to consider cases where the number of particles is large or where the lattice spacing is small. Limits such as these often make explicit the nature of links between models capturing different scales, and, once established, provide a means of improving our understanding, or the models themselves. Finding appropriate variables whose limits illustrate the important connections between models is no easy task, however. This is one area where computer simulation is extremely helpful, as it allows us to see the results of complex dynamics and gather clues regarding the roles of different physical quantities. On the other hand, connections between models enable the development of novel multiscale computational schemes, so understanding can assist computation and vice versa. Some of these ideas are demonstrated in this thesis. The important outcomes of this thesis include: (1) a systematic derivation of the step-flow model of Burton, Cabrera, and Frank, with corrections, from an atomistic solid-on-solid-type models in 1+1 dimensions; (2) the inclusion of an atomistically motivated transport mechanism in an island dynamics model allowing for a more detailed account of mound evolution; and (3) the development of a hybrid discrete-continuum scheme for simulating the relaxation of a faceted crystal mound. Central to all of these modeling and simulation efforts is the presence of steps composed of individual layers of atoms on vicinal crystal surfaces. Consequently, a recurring theme in this research is the observation that mesoscale defects play a crucial role in crystal morphological evolution.

Building an Ontology of Product/Service-Systems: Using a Maritime Case Study to Elicit Classifications and Characteristics

Part 4: Transition Towards Product-Service Systems

Lot production size problem and simulation of urban transport

OBJECTIVES AND STUDY METHOD: There are two subjects in this thesis: “Lot production size for a parallel machine scheduling problem with auxiliary equipment” and “Bus holding for a simulated traffic network”. Although these two themes seem unrelated, the main idea is the optimization of complex systems. The “Lot production size for a parallel machine scheduling problem with auxiliary equipment” deals with a manufacturing setting where sets of pieces form finished products. The aim is to maximize the profit of the finished products. Each piece may be processed in more than one mold. Molds must be mounted on machines with their corresponding installation setup times. The key point of our methodology is to solve the single period lot-sizing decisions for the finished products together with the piece-mold and the mold-machine assignments, relaxing the constraint that a single mold may not be used in two machines at the same time. For the “Bus holding for a simulated traffic network” we deal with One of the most annoying problems in urban bus operations is bus bunching, which happens when two or more buses arrive at a stop nose to tail. Bus bunching reflects an unreliable service that affects transit operations by increasing passenger-waiting times. This work proposes a linear mathematical programming model that establishes bus holding times at certain stops along a transit corridor to avoid bus bunching. Our approach needs real-time input, so we simulate a transit corridor and apply our mathematical model to the data generated. Thus, the inherent variability of a transit system is considered by the simulation, while the optimization model takes into account the key variables and constraints of the bus operation. CONTRIBUTIONS AND CONCLUSIONS: For the “Lot production size for a parallel machine scheduling problem with auxiliary equipment” the relaxation we propose able to find solutions more efficiently, moreover our experimental results show that most of the solutions verify that molds are non-overlapping even if they are installed on several machines. We propose an exact integer linear programming, a Relax&Fix heuristic, and a multistart greedy algorithm to solve this problem. Experimental results on instances based on real-world data show the efficiency of our approaches. The mathematical model and the algorithm for the lot production size problem, showed in this research, can be used for production planners to help in the scheduling of the manufacturing. For the “Bus holding for a simulated traffic network” most of the literature considers quadratic models that minimize passenger-waiting times, but they are harder to solve and therefore difficult to operate by real-time systems. On the other hand, our methodology reduces passenger-waiting times efficiently given our linear programming model, with the characteristic of applying control intervals just every 5 minutes.

Exact solutions for the agricultural and the two-dimensional packing problems

Objectives and study method: The objective of this study is to develop exact algorithms that can be used as management tools for the agricultural production planning and to obtain exact solutions for two of the most well known twodimensional packing problems: the strip packing problem and the bin packing problem. For the agricultural production planning problem we propose a new hierarchical scheme of three stages to improve the current agricultural practices. The objective of the first stage is to delineate rectangular and homogeneous management zones into the farmer’s plots considering the physical and chemical soil properties. This is an important task because the soil properties directly affect the agricultural production planning. The methodology for this stage is based on a new method called “Positions and Covering” that first generates all the possible positions in which the plot can be delineated. Then, we use a mathematical model of linear programming to obtain the optimal physical and chemical management zone delineation of the plot. In the second stage the objective is to determine the optimal crop pattern that maximizes the farmer’s profit taken into account the previous management zones delineation. In this case, the crop pattern is affected by both management zones delineation, physical and chemical. A mixed integer linear programming is used to solve this stage. The objective of the last stage is to determine in real-time the amount of water to irrigate in each crop. This stage takes as input the solution of the crop planning stage, the atmospheric conditions (temperature, radiation, etc.), the humidity level in plots, and the physical management zones of plots, just to name a few. This procedure is made in real-time during each irrigation period. A linear programming is used to solve this problem. A breakthrough happen when we realize that we could propose some adaptations of the P&C methodology to obtain optimal solutions for the two-dimensional packing problem and the strip packing. We empirically show that our methodologies are efficient on instances based on real data for both problems: agricultural and two-dimensional packing problems. Contributions and conclusions: The exact algorithms showed in this study can be used in the making-decision support for agricultural planning and twodimensional packing problems. For the agricultural planning problem, we show that the implementation of the new hierarchical approach can improve the farmer profit between 5.27% until 8.21% through the optimization of the natural resources. An important characteristic of this problem is that the soil properties (physical and chemical) and the real-time factors (climate, humidity level, evapotranspiration, etc.) are incorporated. With respect to the two-dimensional packing problems, one of the main contributions of this study is the fact that we have demonstrate that many of the best solutions founded in literature by others approaches (heuristics approaches) are the optimal solutions. This is very important because some of these solutions were up to now not guarantee to be the optimal solutions.

Biological and chemical strategies for the recovery of precious and rare metals as nanoparticles

Tese de Doutoramento, Ciências do Mar da Terra e do Ambiente, Ramo: Ciências e Tecnologias do Ambiente, Especialidade em Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Cost sharing solutions defined by non-negative eigenvectors

The problem of sharing a cost M among n individuals, identified by some characteristic ci∈R+,ci∈R+, appears in many real situations. Two important proposals on how to share the cost are the egalitarian and the proportional solutions. In different situations a combination of both distributions provides an interesting approach to the cost sharing problem. In this paper we obtain a family of (compromise) solutions associated to the Perron’s eigenvectors of Levinger’s transformations of a characteristics matrix A. This family includes both the egalitarian and proportional solutions, as well as a set of suitable intermediate proposals, which we analyze in some specific contexts, as claims problems and inventory cost games.

Study of extraction conditions for phenolic compounds from Strawberry fruits

Strawberries are an important source of phytochemicals, namely vitamins and phenolic compounds such as anthocyanins and tannins with antioxidant properties [1]. The yield and phenolic content of natural extracts are dependent on the conditions used for extraction [2]. In the present work three different types of extracting solutions (methanol, ethanol:water and aceton:water), two times of extraction (15 and 60 min) and three ratios of solid/solvent (5/25, 5/50 and 5/100 g/mL) were tested in order to evaluate the efficiency of the extraction of phenolic compounds. Phenolic compounds were determined by Folin-Ciocalteu method [3]. Each assay was performed in triplicate. Regarding the extraction solution, it was possible to observe a slight tendency towards a higher efficiency of acetone:water (AcO:H2O, 60:40), but the differences mioght not be statistically significant. A longer time of contact, 60 min as opposed to 15 min, did not show advantages in the yield of extraction. Considering the factors under study, the results obtained showed that volume of extraction solution was the parameter that most influenced the values obtained. Using a higher volume lead to an increase in the amount of phenolic compounds extracted, in a more pronounced way for 15 min of extraction. For a volume of 25 mL the amount of phenolic compounds quantified ranged from 2.13-2.41 mg GAE/g, and increased 30-68% when it was used 50 mL of solution. Using 100 mL of solution, it was extracted twice as double of phenolic compounds. In case of 60 min, the amount of phenolic compounds quantified in samples obtained with 25 mL of solution ranged from 2.32-2.97 mg GAE/g, and increased for 2.43-4.27 mg GAE/g and 3.98-4.68 mg GAE/g when was used 50 and 100 mL, respectively.

Conservation Agriculture and its contribution to the achievement of agri-environmental and economic challenges in Europe

Conservation Agriculture is an ecosystem approach to farming capable of providing solutions for numerous of the agri-environmental concerns in Europe. Certainly, most of the challenges addressed in the Common Agriculture Policy (CAP) could be tackled through Conservation Agriculture (CA). Not only the agri-environmental ones, but also those concerning farmer and rural communities’ prosperity. The optimisation of inputs and similar yields than conventional tillage, make Conservation Agriculture a profitable system compared to the tillage based agriculture. Whereas this sustainable agricultural system was conceived for protecting agrarian soils from its degradation, the numerous collateral benefits that emanate from soil conservation, i.e., climate change mitigation and adaptation, have raised Conservation Agriculture as one of the global emerging agrosciences, being adopted by an increasing number of farmers worldwide, including Europe.

Unbounded solutions for functional problems on the half-line

This paper presents an existence and localization result of unbounded solutions for a second-order differential equation on the half-line with functional boundary conditions. By applying unbounded upper and lower solutions, Green's functions and Schauder fixed point theorem, the existence of at least one solution is shown for the above problem. One example and one application to an Emden-Fowler equation are shown to illustrate our results.

Unbounded solutions for functional problems on the half-line

This paper presents an existence and localization result of unbounded solutions for a second-order differential equation on the half-line with functional boundary conditions. By applying unbounded upper and lower solutions, Green's functions and Schauder fixed point theorem, the existence of at least one solution is shown for the above problem. One example and one application to an Emden-Fowler equation are shown to illustrate our results.